EP0251121A2 - Process for inducing the formation of antibodies - Google Patents

Abstract

Antigenic substances with amino and / or carboxyl groups are maintained while maintaining their electrochemical state, i.e. while maintaining the amino and carboxyl groups, covalently bound to a particulate carrier that is not degradable in the organism of a vertebrate and introduced as an antigen into the organism of a vertebrate, where an immune response of high specificity and long-lasting duration is elicited.

Description

Antibodies are widely used for diagnostic and therapeutic purposes in human and veterinary medicine, as well as for analytical purposes in the field of biochemistry. Sera for the therapy and prophylaxis of viral diseases are classic antibody preparations that have been in use for many decades.

The usual method for producing antibodies is to administer an antigen to the organism of a vertebrate, which produces antibodies as a so-called immune response. These can either be obtained directly from the blood of the vertebrate or one takes antibody-producing cells from the treated organism and develops them further into antibody-producing cell cultures.

Since the antigens in the vertebrate organism are usually broken down rapidly, the antigen usually has to be administered several times until there is a sufficiently strong formation of antibodies.

Antigens have already been bound to particulate carrier substances which are not themselves broken down in the vertebrate organism. In some cases, such carrier-bound antigens can trigger an increased or prolonged immune response.

The antigen can be bound to the carrier by adsorption. For example, viruses, Fab fragments or pollen antigens were adsorptively bound to aluminum hydroxide gels, microcrystalline cellulose, synthetic polymers based on acrylamide, methyl methacrylate or ionic vinyl monomers (cf. US 3,651,213; GT Stevenson, Nature 1974, 247 (5441), pp. 477-478; DE-OS 19 42 196; DE-OS 19 42 161). The adsorptive binding has the advantage that the antigen is not changed by the binding process, but the disadvantage that the binding is reversible, so that the antigen is lost more slowly than in the unbound state, but is inexorably lost.

U. Gröschel-Stewart et.al. had antigen proteins covalently bound to polyacrylamide carriers (U. Groeschel-Stewart et al. Histochemistry 50, 271-279 (1977)) and thus received an immune response in the rabbit organism. The covalent bond attacks amino groups of the antigen to form amide groups. In contrast to the original amino group, the amide group is electrically neutral. The electrochemical state of the antigen is therefore changed by this binding via amide groups. As a result, the immune response is also changed and is less specific for the unbound antigen. Furthermore, the amide bond is not resistant to hydrolysis.

The object of the invention is to trigger the formation of antibodies of increased specificity when the antigen is introduced into the organism of a vertebrate and to further extend the duration of the antibody formation as far as possible.

The object is achieved by the method specified in the claims. The invention makes use of binding processes known per se, in which the carboxyl and amino groups of the antigenic active substance are not so common in the Binding process are included that their electrochemical state is changed. The electrochemical state is caused by the fact that carboxyl and amino groups, depending on the pH or their interaction with charged particles, can change into electrically charged ions, that is to say carboxylate or ammonium ions. This ability is retained in the binding according to the present invention.

As a result of the unchanged electrochemical state of the bound antigenic active substance, an immune response is produced in the vertebrate organism which has a higher specificity than the unbound antigenic active substance than if the immune response had been triggered by an antigenic active substance bound to change the electrochemical state.

There are various possibilities for the covalent binding of antigenic active substances which have amino and / or carboxyl groups to a particulate carrier material which is not degradable in the vertebrate organism, the use of a carrier material constructed in a certain way being of crucial importance.

Carrier materials based on synthetic polymers which contain oxirane groups are preferred. Under mild conditions, the oxirane groups are able to react covalently with the primary or secondary amino groups to form a beta-hydroxyethyl group which occurs as an additional substituent on the amino group, primary in secondary and secondary in tertiary amino groups pass over. The electrochemical state remains practically unchanged. From EP-B 54 249 particles with an average diameter of 0.03 to 10 microns are known, which consist of a crosslinked polymer of glycidyl (meth) acrylate or its copolymer with hydrophilic comonomers, to which immunogenic substances such as antigens or Antibodies that are covalently bound. The use of such immunoparticles to trigger antibody formation in the vertebrate organism is not known. Larger carrier particles for binding biologically active substances are produced according to DE-C 27 22 751 from monomers containing oxirane groups, acrylamide or methacrylamide and / or methylenebis (meth) acrylamide. They have diameters from 5 to 1000 µm.

Another binding method is based on the method described in DE-C 22 63 289. It is particularly suitable for antigenically active substances with a peptide structure, units of glycine, cysteine, cystine, methionine or tryptophan in particular being capable of binding. The binding reaction takes place according to a radical mechanism between a hydrocarbon group of the peptide and a radical-activatable group of the carrier polymer, the amino and carboxyl groups remaining unchanged. Radically activatable groups in this sense are, for example, alkenyl and alkylphenyl groups, preferably allyl and toluyl groups. Activation takes place in the presence of a photosensitizer using ultraviolet light or - if an organic peroxide is additionally present - using visible light in the absence of oxygen.

The binding of antigenic substances with a peptide structure to a synthetic carrier polymer material containing olefinic C = C double bonds is achieved by the process described in DE-C 24 30 128 in the presence of a radical-decomposing compound or a redox system. In contrast to inclusion processes, in which antigenically active substances are incorporated into the matrix of an emerging vinyl polymer without covalent bonding, the process discussed here is carried out in the absence of free-radically polymerizable monomers.

The binding of peptides or other biogenic substances with an antigenic effect to latex-shaped carriers is described in EP-A 65 069. If the methods described therein are used for the purposes of the present invention, those binding-active groups must be selected which generate a covalent bond while maintaining the amino and carboxyl groups of the antigenic active substance. Activated carbon or sulfonic acid groups, which would react with amino groups to form amides, are therefore unsuitable. In addition to the oxirane groups already mentioned, e.g. Haloalkyl and haloalkoyl groups are suitable.

An essential property of the carrier particles is their resistance to degradation in the vertebrate organism. Carriers that perform their carrier function at least as long as the substance bound to them retain their antigenic activity are to be regarded as non-degradable in the sense of the invention. This property is primarily possessed by synthetic vinyl polymers, since they have a continuous carbon chain that cannot be split by the hydrolytic enzymes that are widespread in organisms.

• 1. Monomere mit bindungsaktiven Gruppen, wie
  • a) Oxirangruppen enthaltende Monomere, die mit Aminogruppen des antigen wirksamen Stoffes unter Öffnung des Oxiranringes und Alkylierung des Stickstoffatoms reagieren. Beispiele sind: Glycidylacrylat und -methacrylat, Alkylglycidyläther;
  • b) Halogenalkylgruppen enthaltende Monomere, insbesondere Chloralkyl- oder Bromalkylverbindungen, die mit Aminogruppen unter N-Alkylierung reagieren. Beispiele sind:
    Chloracetoxyalkyl(meth)acrylate,
    Bromalkyl(meth)acrylate,
    Chloressigsäurevinylester;
  • c) Aromatische Gruppen enthaltende Monomere, die unter Einwirkung einer aktivierenden Strahlung mit einer Kohlenwasserstoffgruppierung des antigen wirksamen Stoffes unter Ausbildung einer kovalenten C-C-Bindung reagieren. Dazu gehören: Styrol, Vinyltoluol, Benzylacrylat und -methacrylat;
  • d) Alkenyl-Seitengruppen neben einer radikalisch polymerisierbaren Vinyl- bzw. Vinylidengruppe enthaltende Monomere, die bei Anregung durch eine aktivierende Strahlung oder durch chemisch erzeugte Radikale mit einer Kohlenwasserstoffgruppierung des antigen wirksamen Stoffes unter Ausbildung einer kovalenten C-C-Bindung zu reagieren vermögen. Beispiele für geeignete Monomere sind: Allylacrylat und -methacrylat, Polyallylester mehrbasischer Säuren wie Triallylcyanurat.
• 2. Vernetzende Monomere mit 2 oder mehr radikalisch polymerisierbaren Kohlenstoffdoppelbindungen im Molekül. Die in 1 d erwähnten Monomeren wirken teilweise bei der Polymerisation vernetzend. Im Gegensatz zu den dort genannten Monomeren mit Alkenylgruppen enthalten vernetzende Monomere wenigstens zwei Kohlenstoffdoppelbindungen,die durch benachbarte elektronenanziehende Gruppen aktiviert sind. Beispiele sind Diacrylate oder Dimethacrylate von Glykolen, wie Äthylenglykol, Propylenglykol oder Butylenglykol, Tri- und Tetraacrylate bzw. Tri- und Tetramethacrylate von 3- oder 4-wertigen Alkoholen, wie Trimethylolpropan oder Pentaerythrit; Methylen- bis-acrylamid oder -methacrylamid, Divinylbenzol.
• 3. Hydrophile Monomere, die sich durch eine gewisse Wasserlöslichkeit auszeichnen. Vorzugsweise bilden sie wenigstens 10 %ige wäßrige Lösungen bei 25 Grad Celsius. Dazu gehören Acrylamid, Methacrylamid, Vinylpyrrolidon, Hydroxyalkylester der Acryl- oder Methacrylsäure, vorzugsweise mit 2 bis 4 C-Atomen im Hydroxyalkylrest. Diese Monomeren sind bevorzugt, weil sie elektroneutral sind und den elektrochemischen Zustand des gebunden antigen wirksamen Stoffes nicht beeinflussen. In manchen Fällen können auch ionisierbare Monomere verwendet werden; dazu gehören äthylenisch ungesättigte, polymerisierbare Mono- und Dicarbonsäuren sowie ihre wasserlöslichen Salze, beispielsweise Acryl-, Methacryl-, Itakon-, Malein- oder Fumarsäure. Weiterhin gehören Aminoalkylester und Aminoalkylamide von ungesättigten Carbonsäuren, wie Dialkylaminoalkylester oder Dialkylaminoalkylamide der Acryl- und Methacrylsäure, die vorzugsweise 1 bis 5 C-Atome in den Alkylresten enthalten, zu dieser Gruppe.
  Hohe Anteile an hydrophilen Monomeren machen das Polymer wasserlöslich, was für den Einsatz als partikelförmiges Antigen ungeeignet ist. Daher werden in diesem Falle ausreichende Mengen an vernetzenden Monomeren mitverwendet, um das Polymer wasserunlöslich zu machen. Niedrige Vernetzeranteile, z.B. unter 5 Gew.-%, ergeben im allgemeinen gelartig quellbare Polymere, während hohe Vernetzeranteile nicht quellbare makroporöse Teilchen oder solche mit Hohlperlstruktur ergeben.
• 4. Hydrophobe Monomere, die sich durch eine Wasserlöslichkeit unter 10 % bei 25 Grad Celsius auszeichnen. Sie können beispielsweise angewendet werden, wenn das Trägerpolymer in Latexform hergestellt wird. In manchen Fällen vermögen sie durch hydrophobe Wechselwirkung mit dem antigen wirksamen Stoff dessen Bindung zu fördern. Die unter 1c und 1d genannten Monomeren fallen gleichzeitig in die Gruppe der hydrophoben Monomeren. Weiterhin gehören dazu die Ester von polymerisierbaren ungesättigten Mono- und Dicarbonsäuren mit aliphatischen Alkoholen, die vorzugsweise 1 bis 12, insbesondere 1 bis 4 C-Atome enthalten, beispielsweise Methyl-, Äthyl- ­oder Butyl-acrylat oder -methacrylat, ferner Olefine wie Äthylen oder Propylen, Vinylchlorid, Vinylester gesättigter aliphatischer Carbonsäuren mit 2 bis 10 C-Atomen.

The already mentioned patents describe suitable carrier polymers in detail. Characteristic structural components of these polymers are:

• 1. monomers with bond-active groups, such as
  • a) Monomers containing oxirane groups, which react with amino groups of the antigenic active substance to open the oxirane ring and alkylate the nitrogen atom. Examples are: glycidyl acrylate and methacrylate, alkyl glycidyl ether;
  • b) monomers containing haloalkyl groups, in particular chloroalkyl or bromoalkyl compounds, which react with amino groups with N-alkylation. Examples are:
    Chloroacetoxyalkyl (meth) acrylates,
    Bromoalkyl (meth) acrylates,
    Vinyl chloroacetate;
  • c) Monomers containing aromatic groups, which react under the action of activating radiation with a hydrocarbon grouping of the antigenic active substance to form a covalent CC bond. These include: styrene, vinyl toluene, benzyl acrylate and methacrylate;
  • d) alkenyl side groups in addition to a radically polymerizable vinyl or vinylidene-containing monomers which, when excited by activating radiation or by chemically generated radicals, are able to react with a hydrocarbon grouping of the antigenic active substance to form a covalent CC bond. Examples of suitable monomers are: allyl acrylate and methacrylate, polyallyl esters of polybasic acids such as triallyl cyanurate.
• 2. Crosslinking monomers with 2 or more radical polymerizable carbon double bonds in the molecule. Some of the monomers mentioned in 1 d have a crosslinking action during the polymerization. In contrast to the monomers mentioned there with alkenyl groups, crosslinking monomers contain at least two carbon double bonds which are activated by adjacent electron-accepting groups. Examples are diacrylates or dimethacrylates of glycols, such as ethylene glycol, propylene glycol or butylene glycol, tri- and tetraacrylates or tri- and tetramethacrylates of trihydric or tetravalent alcohols, such as trimethylolpropane or pentaerythritol; Methylene bis-acrylamide or methacrylamide, divinylbenzene.
• 3. Hydrophilic monomers, which are characterized by a certain water solubility. They preferably form at least 10% aqueous solutions at 25 degrees Celsius. These include acrylamide, methacrylamide, vinylpyrrolidone, hydroxyalkyl esters of acrylic or methacrylic acid, preferably with 2 to 4 carbon atoms in the hydroxyalkyl radical. These monomers are preferred because they are electroneutral and do not affect the electrochemical state of the bound antigenic active substance. In some cases, ionizable monomers can also be used; these include ethylenically unsaturated, polymerizable mono- and dicarboxylic acids and their water-soluble salts, for example acrylic, methacrylic, itaconic, maleic or fumaric acid. This group also includes aminoalkyl esters and aminoalkylamides of unsaturated carboxylic acids, such as dialkylaminoalkyl esters or dialkylaminoalkylamides of acrylic and methacrylic acid, which preferably contain 1 to 5 carbon atoms in the alkyl radicals.
  High levels of hydrophilic monomers make the polymer water-soluble, which is unsuitable for use as a particulate antigen. In this case, therefore, sufficient amounts of crosslinking monomers are used to render the polymer water-insoluble. Low levels of crosslinker, for example below 5% by weight, generally result in gel-like swellable polymers, while high levels of crosslinker give non-swellable macroporous particles or those with a hollow bead structure.
• 4. Hydrophobic monomers, which are characterized by water solubility below 10% at 25 degrees Celsius. For example, they can be used when the carrier polymer is made in latex form. In some cases they can promote the binding of the antigenic active substance through hydrophobic interaction. The monomers mentioned under 1c and 1d fall simultaneously into the group of hydrophobic monomers. These also include the esters of polymerizable unsaturated mono- and dicarboxylic acids with aliphatic alcohols, which preferably contain 1 to 12, in particular 1 to 4, carbon atoms, for example methyl, ethyl or butyl acrylate or methacrylate, furthermore olefins such as ethylene or Propylene, vinyl chloride, vinyl esters of saturated aliphatic carboxylic acids with 2 to 10 carbon atoms.

• 1) Bindungsaktive Monomere: 1 bis 80 Gew.-% vorzugsweise 5 bis 50 Gew.-%
• 2) Vernetzende Monomere: 0 bis 50 Gew.-%, vorzugsweise 0,01 bis 30 Gew.-%
• 3) Hydrophile Monomere 0 bis 99 Gew.-%, vorzugsweise 20 bis 95 Gew.-%
• 4) Hydrophobe Monomere 0 bis 50 Gew.-%, vorzugsweise 0 bis 30 Gew.-%.

The relative proportions of the monomers involved depend on their function, ie the need required for their action. Unless suitable monomer proportions are already specified in the above-mentioned patents, the following ranges can serve as guide values.

• 1) Binding-active monomers: 1 to 80% by weight, preferably 5 to 50% by weight
• 2) Crosslinking monomers: 0 to 50% by weight, preferably 0.01 to 30% by weight
• 3) hydrophilic monomers 0 to 99% by weight, preferably 20 to 95% by weight
• 4) Hydrophobic monomers 0 to 50% by weight, preferably 0 to 30% by weight.

For the desired effect of the carrier polymers, it is sufficient if the outermost layer of the polymer particles, and in the case of porous or macroporous polymer particles also the pore walls, consist of the polymer material described. Below this can be a core made of another material, for example an inorganic carrier body or a seed latex core made of a hydrophobic polymer such as polystyrene, poly (meth) acrylic esters and the like. the like

The size of the particles of the carrier polymer depends on the type of introduction into the vertebrate organism. If the particulate antigen is introduced into the bloodstream, the particle size is preferably in the range from 0.01 to 10 μm. When embedded in the tissue, larger particles, for example up to 100 µm, can also be used. Carrier particles in the range from 0.01 to 2 μm are advantageously produced by emulsion polymerization. Particles of the order of 1 to 50 μm are accessible by precipitation polymerization in a medium which is a solvent for the monomers and a non-solvent for the polymers. The suspension or bead polymerization in a medium which does not dissolve either the monomer phase or the polymer particles leads to particles between 2 and 100 μm. Coarser particles or bulk polymers can be produced by impact mills, homogenizers and the like. be crushed into particles in these areas. The quantitative ratio of antigenic active substance and carrier material can be in a wide range. A high effectiveness is generally achieved in the range between 1:10 and 1: 1000.

Antigenic active substances are understood to mean all substances which are able to trigger an immune reaction in the organism of a vertebrate. This includes practically all foreign, natural or synthetic macromolecules with molecular weights over 2000, as well as surface structures of foreign particles, such as active or inactivated bacteria or viruses, which are made up of such macromolecules. The invention aims to use such antigenic active substances which contain one or more carboxyl and / or amino groups. The process can be carried out in the same way with antigenic active substances without carboxyl and amino groups, but the advantage according to the invention, which is based on the maintenance of the electrochemical state, then lapses. The amino or carboxyl groups are the same as the ammonium or carboxylate groups resulting from salt formation. The amino groups can be primary, secondary or tertiary. Although the presence of at least one amino group and one carboxyl group is the rule, antigenic active substances which contain only one or more amino groups but no carboxyl group, or one or more carboxyl groups but no amino group, are within the scope of the invention.

The antigenic active substances within the meaning of the invention also include haptens; these are substances that are not alone an antigen in the unbound state, but only become an antigen after binding to a carrier. In practice, the question of whether a certain substance also acts as an antigen when it is unbound is difficult to decide, because in some cases the immune response is only triggered after the substance has bound to an undissolved substance in the body. There is therefore no limit fundamental type for the molecular weight or the size of the antigenic active substance. The substance can be soluble or insoluble in water and in the latter case must be soluble in a liquid suitable for the covalent bond.

An outstanding group of antigenic active substances are those with a protein or peptide structure, which can occur alone or in combination with non-protein or peptide-like components. Peptides and proteins usually contain an amino and a carboxyl group as end groups. Other groups of practical importance are polysaccharides and nucleic acids. If they do not themselves carry carboxyl or amino groups, they can be linked to substances containing such groups. Penicillins, tetracylines and digoxin are examples of haptens.

The vertebrates treated according to the invention are preferably warm-blooded animals. Suitable are e.g. Cattle, sheep, goats, horses, pigs, dogs, cats, rabbits, rats and mice among the mammals and chickens, ducks, geese, turkeys, pigeons among the birds. The invention is also applicable to cold-blooded animals, such as fish and reptiles.

For application, the particulate antigen can be suspended in an isotonic solution and injected into the bloodstream or stored in the skin or connective tissue. The immune response can usually be observed after an incubation period of 3 to 20 days. As a rule, the one-off is sufficient Application of the antigen. The removal of antibody-containing blood or serum or of antibody-producing cells for the establishment of cell cultures takes place in a manner known per se, such as after triggering the immune response with antigen not bound to carrier particles.

Example:

2 g of a powdery carrier material redispersible to particles of about 1 micron diameter (commercial product EUPERGIT ClZ, Röhm GmbH, Darmstadt), which contains binding-active oxirane groups (produced according to DE 3l 16995; or US Patent Appl. Serial No. 930 023 ) are suspended in 4 ml of a buffer solution of pH 7.3 (1.0 M Na phosphate + 0.15 NaCl) and 20 mg bovine serum albumin ("BSA", manufacturer Miles Lab, Lot No. 63, Code No.8l- 00l) added. The suspension is left to stand at room temperature for 72 hours. Then it is washed several times with water and centrifuged. The Bradford protein test (carried out using a Bio-Rad test kit) shows a content of 4.4 mg of covalently bound BSA per gram of carrier material.

455 mg of the immobilizate obtained, containing 2.0 mg of BSA, are mixed with 1 ml of incomplete Freund's adjuvant (an auxiliary which is generally known in immunochemistry) and suspended in ultrasound for 40 minutes to form a water-in-oil emulsion. The suspension is subcutaneously administered to a rabbit in 10 individual doses. The whole treatment is repeated after three weeks to enhance the immune effect. After a further four weeks, 10 ml of blood are withdrawn from the ear vein from the treated rabbit. The blood drawn is left in the refrigerator for one hour at 37 ° C. and for a further 36 hours. Then the anti-serum is separated from the blood cake in the usual way.

The antibody concentration in the antiserum can easily be measured with the capillary test. For this purpose, the antigen, namely BSA, is dissolved to a concentration of 1 mg / ml in PBS (phosphate buffer, saline, pH 7.3). The solution is filled into a glass capillary at a filling height of 3 cm. Then hold the same amount of the antiserum obtained from the rabbit blood into the same capillary by holding the capillary at an angle and seal the lower end of the capillary with putty. The capillary is left at 37 ° C for 1 hour and another 48 hours at 5-8 ° C and the height of the precipitate deposited is measured at the lower end of the capillary. A precipitate height of 12 mm is measured. A precipitate height of 10 mm corresponds to an antibody concentration of about 1 mg / ml. This results in an antibody concentration of 1.2 mg / ml in the antiserum.

For comparison, the blood serum of an untreated rabbit is treated in the same way with the antiserum. No precipitate forms in the capillary at all.

In a further experiment, only the immobilized BSA (without incomplete Freund's adjuvant) is administered subcutaneously to a rabbit and the treatment is repeated as described above. The immunoassay only shows a precipitate height of 4 mm, which corresponds to an antibody concentration of 0.4 mg / ml. This results in the increased effectiveness of the immobilized BSA in the presence of incomplete Freund's adjuvant.

The corresponding antisera can be prepared in a corresponding manner when insulin or thyroid stimulating hormone ("TSH") is used instead of BSA. They can be used for diagnostic purposes to determine insulin or TSH.

Claims (13)

1. A method for triggering antibody formation in the organism of a vertebrate, wherein an antigenic active substance containing amino and / or carboxyl groups is immobilized by covalent binding to a particulate carrier which is not degradable in the organism of the vertebrate to form a particulate antigen and the antigen is immobilized into the vertebrate organism,

characterized,

that the antigenic active substance is covalently bound to the carrier while maintaining its amino and carboxyl groups. 2. The method according to claim 1, characterized in that one uses a carrier containing oxirane groups and the antigenic active substance is immobilized by reaction with the oxirane groups. 3. The method according to claim 1, characterized in that one uses a carrier containing alkenyl groups and / or alkylphenyl groups and the antigenic active substance in the presence of a photosensitizer by means of ultraviolet light or - in the additional presence of an organic peroxide - by means of visible light in the absence of oxygen covalently immobilized on the carrier. 4. The method according to claim 1, characterized in that a carrier with olefinic C = C double bonds is used and the antigenic active substance is covalently bound to the carrier in the presence of a compound which decomposes to free radicals or a redox system in the absence of free-radically polymerizable monomers. 5. The method according to any one of claims 1 to 4, characterized in that one uses a carrier to 1) 1-88% by weight of binding-active monomers which contain an oxirane group, a haloalkyl group, an aromatic group or - in addition to a polymerizable vinyl or vinylidene group - an alkenyl side group, 2) 0-50% by weight of crosslinking monomers with two or more free-radically polymerizable carbon double bonds in the molecule, 3) 0 to 99% by weight of hydrophilic monomers which form at least 10% aqueous solutions at 25 degrees Celsius, 4) 0 - 50 wt .-% of hydrophobic monomers that form less than 10% aqueous solutions at 25 degrees Celsius is constructed. 6. The method according to claims 1 to 5, characterized in that the antigenic active substance is bound to a particulate carrier with an average particle diameter between 0.01 and 100 microns. 7. The method according to claim 6, characterized in that the antigenic substance is bound to a present as an aqueous latex carrier with a particle size between 0.01 and 2 microns. 8. The method according to claims 1 to 7, characterized in that a substance with a protein or peptide structure is used as the antigenic active substance. 9. The method according to claims 1 to 7, characterized in that a hapten containing one or more amino and / or carboxyl groups is used as the antigenic substance. 10. The method according to claims 1 to 9, characterized in that the particulate antigen is introduced into the organism of a warm-blooded vertebrate. 11. The method according to claim l0, characterized in that the particulate antigen is introduced subcutaneously into the organism of the vertebrate. 12. The method according to claim l0, characterized in that the particulate antigen is introduced in the form of a water-in-oil emulsion in the organism of the vertebrate.